Therapeutic proteins which are generally administered by intravenous injection may be immunogenic, relatively water insoluble, and may have a short in vivo half-life. The pharmacokinetics of the particular protein will govern both the efficacy and duration of effect of the drug. It has become of major importance therefore to reduce the rate of clearance of the protein so that prolonged action can be achieved. This can be accomplished by conjugation of a therapeutic polypeptide with a polymer such as polyethylene glycol (PEG). The effective molecular volume of the protein is thus increased and glomerular filtration is either avoided or inhibited (Brenner et al., (1978) Am. J. Physiol., 234, F455). By increasing the molecular volume and by masking potential epitope sites, modification of a therapeutic polypeptide with a polymer such as polyethylene glycol (PEG) has been shown to be efficacious in reducing both the rate of clearance as well as the antigenicity of the protein. Reduced proteolysis, increased water solubility, reduced renal clearance, and steric hindrance to receptor-mediated clearance are a number of mechanisms by which the attachment of a PEG polymer to the backbone of a polypeptide may prove beneficial in enhancing the pharmacokinetic properties of the drug. Thus Davis et al., U.S. Pat. No. 4,129,337 discloses conjugating PEG to proteins such as enzymes and insulin to produce a less immunogenic product while retaining a substantial proportion of the biological activity. PEG modification requires activation of the PEG polymer which is accomplished by the introduction of an electrophilic center. The PEG reagent is now susceptible to nucleophilic attack, predominantly by the nucleophilic epsilon-amino group of a lysyl residue or by a free sulfhydryl moiety when available. Because of the number of surface lysines present in most proteins, the pegylation process can result in random attachments leading to mixtures which are difficult to purify and which may not be desirable for pharmaceutical use. When however a free sulfhydryl group is available for conjugation, the pegylation process will result in the site specific introduction of the PEG polymer. There are a large variety of active PEGs which have been developed for the covalent modification of proteins via the formation of a linking group between PEG and protein (see for example Zalipsky, et al., and Harris et. al., in: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21 and 22). Some of these reagents however, are to various degrees, unstable in the aqueous medium in which the pegylation reaction occurs. In addition, the conjugation process often results in the loss of in vitro biological activity which is due to several factors foremost of which being a steric interaction with the protein's active sites. A desired property therefore of a new pegylating reagent would be one that is not susceptible to rapid degradation in an aqueous medium and one which may be employed to affect the site specific modification of a protein. Cross-linking derivatives such as a PEG dialdehyde and a PEG polymer which has an aldehyde function at the alpha position and a sulfydryl specific group at the omega position may be considered as such reagents. For site specific N-terminal reductive amination, see Pepinsky et al., (2001) JPET, 297, 1059 (Interferon-•-1a) and U.S. Pat. No. 5,824,784 (1998) to Kinstler et al., (G-CSF). The use of a PEG-aldehyde for the reductive amination of a protein utilizing other available nucleophilic amino groups, is described in U.S. Pat. No. 4,002,531 (1977) to Royer, in EPO 154 316, by Wieder et al., (1979) J. Biol. Chem. 254, 12579, and Chamow et al., (1994) Bioconjugate Chem. 5, 133. For the site specific pegylation of a protein via a free sulfhydryl group see Goodson et al., Bio/Technology (1990) 8, 343.
As in the case of pegylation with monofunctional PEG reagents, the cross-linking of proteins with a PEG polymer may be employed to enhance the stability of the protein as well as increase the effective molecular volume of those proteins that may otherwise be susceptible to glomerular filtration and therefore rapid clearance from the circulation. Cross-linking reagents have been found in general to have the same type and degree of reactivity as the analogous monofunctional reagent although the length and flexibility of the cross-link may vary greatly. Cross-linking may involve the coupling of two similar or diverse molecular components such as proteins, peptides, or pharmaceuticals which can provide in a single molecular entity two different activities (see for example Saleh et al., (2000) J. Clin. Oncol. 18, 2282).
An example of intramolecular cross-linking is described in the case of hemoglobin, where the cross-bridging process has been used in an attempt to prevent the dissociation of the tetramers into their constituent •,• dimers (Zhang et al. (1989) Biochem. Biophys. Res. Commun. 163, 733; Manjula et al. (2000) J. Biol. Chem. 275, 5527).